الفهرس 
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Abstract
The present investigation was carried out at the Experimental Farm and the Tissue Culture Laboratory of the Department of Vegetable Crops, and at the Biotechnology Laboratory of the Genetics Department, Faculty of Agriculture, Assiut University, Assiut, Egypt, during the period from 2015 to 2019.
The investigation aimed to study the following objectives
I- Assess the variability of important agromorphological traits along with the indicate of variability i.e. genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV), heritability in broad sense and genetic advance as a percent of mean.
II- Study the genetic relationships among garlic genotypes based on agromorphological traits.
III- Study the genetic diversity among garlic genotypes based on different systems of molecular markers.
IV- Study tissue culture response and plant regeneration in garlic (Allium sativum L.)
V- Detect somaclonal variation in regenerated garlic plants as revealed by the analysis of molecular markers.
VI- Assess the possibility of selecting salt-tolerant garlic plants via tissue culture method.
VII- And to study the changes in gene expression under salinity stress using protein and isozymes analysis.
Five genotypes of garlic (Allium sativum L.) were used in the present investigation. In the first part of the present investigation, the performance of these genotypes during two successive winter seasons of 2015/2016 and 2016/2017 was evaluated for nine characters. These characters are; plant weight (PW, gm), plant height (PH, cm), bulb diameter (HD, cm), bulb weight (HW, gm), neck diameter (ND), number of Leaves/plant (LNo), bulbing ratio (BR), cloves number (CNo), and cloves weight (CW, gm). For each trait the genetic, environmental, and phenotypic variances, phenotypic and genotypic coefficients of variation (PCV and GCV), and broad-sense heritability (h2bs) were further estimated from the ANOVA mean squares. The expected genetic advance (GA) was determined at 5% and 20% intensity of selection. The simple correlation coefficients between all studied characters were also determined. In the second part, the interrelationship between the tested genotypes was studied using phylogenetic tree analysis based on agro-morphological characters. The third part was conducted to evaluate the response of these genotypes for tissue culture. In the fourth part of the present investigation, different systems of molecular markers (seven ISSR and nine SRAP markers) were used to study genetic diversity among the five garlic genotypes and to determine the correlations among agro-morphological traits, tissue culture traits, and molecular marker systems. In the fifth part of the present investigation, molecular analysis was used to study the somaclonal variation among plants regenerated from the five garlic genotypes. The sixth part was conducted to evaluate the genetic potential of these genotypes through artificially created salinity stress by NaCl in in-vitro conditions and to study the changes in gene expression under salinity stress.
The results obtained could be summarized as follow
I- Genetic analysis of agro-morphological characters
1. Highly significant differences were found between the tested genotypes in all studied traits.
2. The genotypic component (δ2g) was greater than the environmental component (δ2e) in all studied traits.
3. The phenotypic and genotypic coefficients of variation (PCV and GCV) were high for plant weight (PW), bulb weight (HW), cloves number (CNo), leaves number (LNo) and cloves weight (CW), whereas plant height (PH) and neck diameter (ND) exhibited low values.
4. High heritability estimate associated with high genetic advance were observed for plant weight (PW) and bulb weight (HW) reflecting the preponderance of additive gene action indicating that direct selection could be used to improve these traits.
5. Heritability estimate was high and was associated with moderate genetic advance observed for cloves number (CNo). This indicated the presence of additive and non-additive gene action and suggesting that these traits can be improved by mass selection or other breeding methods based on progeny testing.
6. High heritability estimate associated with low genetic advance was observed for neck diameter (ND), leaves number (LNo), bulbing ratio (BR), and cloves weight (CW). These results indicate that these traits are controlled by non-additive gene action and could be improved through hybridization and hybrid vigor.
7. Moderate heritability associated with low genetic advance was observed for plant height (PH), bulb diameter (HD), which indicate that the influence of error variance on these traits was high and suggest that indirect selection of other secondary traits may be feasible to improve them.
8. Plant weight showed significant positive correlation with plant height (PL), bulb diameter (HD), bulb weight (HW), neck diameter (ND), leaves number (LNo) and bulbing ratio (BR). This indicates that the improvement in any of these traits might result in a positive response of the plant weight in garlic genotypes.
9. Plant height also showed significant positive correlations with bulb diameter (HD), bulb weight (HW), leaves number (LNo), and cloves weight (CW). These indicate that the improvement in any of these traits might result in a positive response of the plant height in garlic genotypes.
10. Bulb diameter (HD) showed a significant positive correlation with bulb weight (HW) and cloves number (CNo) and a negative correlation with the bulbing ratio (BR).
11. Cloves number (CNo) showed a significant negative correlation with the bulbing ratio (BR), leaves number (LNo) and cloves weight (CW).
Genetic relationships among garlic genotypes based on agro-morphological traits
1. The Euclidean distance among all genotypes was 3.07 to 5.52 with an average of 4.91.
2. The dendrogram distinguished five genotypes of garlic into two main clusters. Cluster-1 included one genotype G1 which was separated in a single branch from the other genotypes, reflecting a relatively longer genetic distance from the other varieties. Cluster-2 comprised of the remaining four genotypes and was further divided into two sub-clusters.
II- Tissue culture response
1. Calluses, shoots and roots were formed from all genotypes on all tested concentrations of growth hormones. While, the percentage of callus formation, regeneration rate, number of shoots/explant and percentage of roots were depending upon the genotype and concentration of the growth hormones. The differences between the tested genotypes were highly significant for all studied tissue culture traits.
2. The genotype G2 followed by G1 and G3 were highly responsive for in vitro culture while the two genotypes G4 and G5 were less responsive for in vitro culture.
3. Callus induction medium supplemented with 4 mg/L 2,4-D was the best medium for callus induction for all garlic genotypes while, the regeneration medium containing 0.1 mg IAA + 2 mg/L kinetin was the best for plant regeneration from garlic genotypes.
4. The dendrogram generated from the standardized tissue culture data separated the five garlic genotypes into two clusters; the first cluster comprised three garlic genotypes (G1, G2 and G3) which were highly responsive for tissue culture. The second cluster comprised the two other garlic genotypes (G4 and G5) which were the less responsive in tissue culture generative ability.
III- Genetic relationship among garlic genotypes based on different systems of molecular markers.
1. Two molecular marker systems (seven ISSR primers and nine SRAP primer combinations) were used in the present investigation to study the genetic diversity among the five garlic genotypes.
2. The two molecular markers system generated a total of 191 DNA fragments from all genotypes with an average 11.94 bands/primers.
3. Out of 191 DNA fragments, 107 (56.02%) showed polymorphism and 84 (43.98%) were common bands. This indicates the existence of high relatively genetic diversity among investigated garlic genotypes.
4. The genotype G3 displayed the highest number of DNA fragments (162 bands), while the genotype Egaseed 2 (G5) revealed the least number of bands (130 bands).
5. Both molecular marker systems generated unique DNA bands with different sizes, which could be used as specific marker for a particular genotype.
6. Based on the combined data of ISSR and SRAP markers, the genetic similarity ranged from 0.709 between G2 and G4 to 0.932 between G4 and G5.
7. The dendrogram based on combined ISSR and SRAP data grouped the five genotypes into two main clusters.
8. Highly significant positive correlations were found between the two molecular marker systems and tissue culture traits. In contrast, significant negative correlations were found between agro-morphological traits and each of the tissue culture traits and molecular marker systems.
IV- Somaclonal variation among plants regenerated from the five garlic genotypes (donor parents).
1. Three ISSR primers and three SRAP primer combinations (which gave reproducible and high polymorphic bands among the five garlic genotypes) were used to detect the somaclonal variation among the regenerated plants.
2. Both marker systems generated a total of 78 bands across 50 somaclones and five donor parents.
3. Of 78 DNA fragments, 76 (97.44%) bands were polymorphic and 2 (2.56%) bands were common between all genotypes.
4. The percentage of polymorphism across all primers ranged from maximum 100% (HB08, HB15, SRAP2, SRAP-4, and SRAP-9) to minimum 81.82% (HB10).
5. For donor parent Balady (G1), only two regenerated plants were identical with their donor parent and 8 (80.0%) regenerated plants (somaclones) were varied from donor parent in DNA locus ranged from 8 to 16 loci at genome level.
6. For donor parent Chinese (G2), Seven (70.0%) of regenerated plants (somaclones) were unstable at DNA level and exhibited genetic changes from the donor parent in different loci of genome ranged from 8 loci to 12 loci.
7. For donor parent Sids-40 (G3), 10 of the regenerated plants screened, only 5 (50.0%) plants exhibited genetic change compared with donor parent. These variations at DNA level ranged from 10 to 12 loci.
8. For donor parent Egaseed-1 (G4), all in vitro regenerated plants from Egaseed-1 genotype exhibited genetic change at DNA level in different loci ranged from 7 to 13 loci.
9. For donor parent Egaseed-2 (G5), out of 10 in vitro regenerated plants from the donor parent Egaseed-2, only three plants (somaclones) exhibited genetic change at DNA level in different loci ranged from 10 to 15 loci.
10. The genetic similarity among all somaclones and their donor plants ranged from minimum 0.447 to maximum 1.0 with an average of 0.724.
11. Egaseed-2 (G5) was more stable under in vitro culture conditions in this study, followed by Sids-40 (G3) while Egaseed-2 (G4) was less stable under the same in vitro culture conditions followed by Balady (G1) and Chinese (G2) genotypes.
V- In vitro selection for salinity stress and alteration in gene expression under salt stress.
1. Five different concentrations of NaCl were used to study in vitro response of garlic genotypes for salinity stress based on easily measurable traits. These traits included percentage of callus formation, percentage of shoot formation, number of shoots/explant, percentage of root formation, callus fresh weight, callus dry weight, shoot length, shoot fresh weight, and shoot dry weight.
2. The differences between the genotypes, NaCl concentrations, and the interaction between genotype and concentration were highly significant. This indicate that the response to salt stress depends on the genotype and concentration of the NaCl.
3. Generally, the results revealed that the development of callus and its differentiation were decreased with the increment of NaCl level from 25 to 120 mM. At higher concentrations, almost all cultured explants turned brown and the few that developed calli failed to regenerate shoots. Therefore, it was decided to use the concentration of 120 mM NaCl as a selection stress (~10% response of the control) to enhance salt tolerance in garlic
4. from the five garlic genotypes, 1250 explants were cultured under selection conditions and 750 explants cultured on control medium.
5. Callus formation and shoot regeneration varied from one cultivar to another and from control medium to the salt stressed medium.
6. In general, the percentage of callus formation was sharply reduced from 75.8% in salt-free medium to 18.08% in salt stressed medium (120 mM NaCl),
7. Number of shoots/explants was sharply reduced from 14.58 shoots in salt-free medium to 7.61 shoots in salt stressed medium. These results indicate that the presence of sodium chloride in the culture medium may inhibit the regeneration process in garlic.
8. The analysis of variance revealed highly significant differences between all genotypes as well as between the two concentrations of NaCl.
9. The selected plants exceeded their donor parents and the unselected plants in growth on salinized medium.
10. Results indicated that salinity tolerance was enhanced in the selected plants as compared to the donor parents and unselected plants. However, this enhancement varied from one genotype to another.
11. The maximum enhancement was observed in plants selected from G3 (93.26% of the control) followed by G1 (73.78%) and G2 (58.24%). While those selected from G5 showed the lowest enhancement (34.8% of the control) of growth under salinity stress.
12. The results showed that salinity-stress induced new protein bands in all tested genotypes, as compared to their control treatment. Most of these newly synthesized proteins were of low molecular weights and differed from one genotype to another. These polypeptides may be involved in the mechanism(s) of salt tolerance in garlic. Also, the results revealed that salinity causes induction of isozyme bands, depending on the nature of the isozymes and the tested genotypes.